This renewal proposal continues the applicant's investigation of mechanisms that lead to chronic pain following peripheral nerve damage. Based on data derived from earlier funding periods, he proposes three linked hypotheses concerning the sequence of events following nerve injury: (1) release of substance P (sP) at the injury site acts on NK1 receptors to induce production of tumor necrosis factor alpha (TNF) via modulation of a PKC signal transduction pathway; (2) Both sP and TNF influence expression of endothelial cell adhesion molecules (VCAM-1, ICAM-1, E-selectin) that control macrophage invasion into endoneurial space and development of Wallerian degeneration; (3) Increased retrograde axonal transport of receptor-bound sP and TNF to DRG cells upregulates their expression in DRG cells and leads to aberrant electrophysiologic activity in sensory neurons. The role of sP will be investigated in experiments to correlate the temporal course of changes in substance P and TNF with the development of neuropathy, to attempt to mimic nerve injury by injecting substance P and measuring production of TNF, and to attempt to block various steps in the process by substance P antagonists, a PKC antagonist, TNF blocking proteins, and antisense TNF mRNA. The role of endothelial cell adhesion molecules will be investigated by using antisense VCAM-1, ICAM-1 and E- selectin sequences to block upregulation. The consequences of increased sP and TNF in dorsal root ganglia will be determined by characterizing the axonal transport of these factors and their receptors following injury, measuring the upregulation of corresponding mRNA and density of associated protein in DRG cells, and characterizing the temporal changes in electrophysiological activity in DRG neurons following nerve injury. Studies will be carried out primarily in the Bennett and Xie model of chronic constriction injury of rat sciatic nerve, with some studies also in a less severe partial freeze injury model developed by the investigator. Variables to be collected include behavior, histology, immunohistochemical localization of proteins, and ELISA for quantification. Electrophysiology of spontaneous activity in DRG neurons will use extracellular recordings from single and multiple units in the sensory root.